Abstract Type 2 diabetes afflicts nearly 26 million Americans and causes a larger economic loss than all cancers combined. It starts as insulin resistance, but ultimately the pancreatic Beta-cells that make insulin fail, resulting in overt diabetes. Failure is due in part to aggregation of the hormone known as the human islet amyloid polypeptide (hIAPP or amylin) into amyloid plaques that occupy up to 80% of the islet space. Surprisingly, the amyloid fibers themselves are less cytotoxic than are oligomers of hIAPP. It is unknown how these oligomers impair Beta-cells, but they could interfere with receptor mediated processes or permeabolize the membrane. As a result, there is much interest in understanding the mechanism by which hIAPP aggregates, because the aggregation pathway dictates the structures and populations of these cytotoxic intermediates. However, very little structural information exists about intermediates because standard structural biology tools are difficult to apply to aggregated proteins, let alone kinetically evolving and membrane associated proteins. In the last grant period, we made a technological advance that allowed us to collect 2D IR spectra on-the-fly and thereby monitor the kinetics of hIAPP aggregation. We coupled our spectroscopy with [13]C/[18]O isotope labeling to obtain residue specific structural resolution. In doing so, we made an important discovery: the FGAIL region of hIAPP forms a parallel Beta-sheet intermediate before breaking into the disordered loop of the fiber. This disordering causes a large barrier in the free energy pathway which dictates the kinetics of fiber formation and results in a long lifetime for the intermediate. Our data suggests that this FGAIL intermediate is the oligomeric species currently being sought to explain hIAPP toxicity. It may also be the key to a theo1y for why some species contract type 2 diabetes but not others - a theory used to design drugs to treat type 2 diabetes. Specific Aim 1 will refine the structure of this intermediate and use in vivo assays to test its cytotoxicity. Specific Aim 2 will test if the IAPP from other species also populates this intermediate. Finally, Specific Aim 3 utilizes the capability of 20 IR spectroscopy for studying membrane peptide structure and kinetics. We will map the structure of hIAPP with residue-level specificity as it aggregates on membrane vesicles to identify possible cytotoxic structures. Elucidating the aggregation pathways of hIAPP will help understand Beta-cell failure that ultimately causes overt type 2 diabetes as well as help in the development of hormone replacement therapies. The structures and kinetics that we will obtain will provide a detailed characterization of hIAPP aggregation that is not currently possible with any other technique.?